p40 molecule regulates NK cell activation mediated by NK ... - CiteSeerX

International Immunology, Vol. 9, No. 9, pp. 1271–1279

© 1997 Oxford University Press

p40 molecule regulates NK cell activation mediated by NK receptors for HLA class I antigens and TCR-mediated triggering of T lymphocytes Alessandro Poggi1, Elena Tomasello1, Valentino Revello1, Luca Nanni2, Paola Costa1 and Lorenzo Moretta1,2 1Istituto 2Istituto

Nazionale per la Ricerca sul Cancro e Centro di Biotecnologie Avanzate-IST, 16132 Genova, Italy di Patologia Generale, Universita` degli Studi di Genova, 16132 Genova, Italy

Keywords: activating NKR, HLA-class I, NK cells, regulation, TCR activation

Abstract p40 was previously described as a regulatory molecule capable of inhibiting both the natural and the CD16-mediated cytotoxicity of NK cells. In this study, we analyze the effect of p40 molecule engagement on the NK cell triggering induced by activating HLA class I-specific NK receptors (NKR) or on TCRαβ-mediated T cell activation. CD3–CD16F NK cell clones expressing activating NKR (either CD94 or p50) were analyzed in a redirected killing assay using P815 target cells and appropriate mAb. A strong target cell lysis was detected in the presence of anti-NKR or anti-CD16 mAb alone. Addition of anti-p40 mAb resulted in a strong inhibition of both anti-NKR or anti-CD16 mAb-induced cytolysis. mAb specific for either CD45 or lymphocyte function associated antigen-1 did not exert any inhibitory effect in the same experimental system. Free intracellular calcium ([Ca2F]i) increase induced by mAb cross-linking of activating CD94 or p50 was inhibited by simultaneous engagement of p40 molecules, but not of other NK surface molecules including CD44 and CD56. In addition, cross-linking of p40 molecules strongly inhibited the CD94-induced tumor necrosis factor-α and IFN-γ production. Analysis of TCR αβ or γδ T cell clones revealed that the engagement of p40 molecules, using specific mAb, induced some degree of inhibition only on anti-Vβ (but not anti-Vδ or anti-CD3) mAb-induced cytotoxicity. On the other hand, the p40 molecule engagement prevented T cell proliferation induced by either anti-Vβ8 or anti-Vδ2 mAb. A similar inhibitory effect was found on the IL-2-induced NK cell proliferation. Taken together, our present findings suggest that p40 may play a role in the regulation of NK and T lymphocyte activation and proliferation. Introduction In recent years numerous studies have been focused on surface molecules involved in down-regulation of lymphocyte function. Thus FcγRIIB was found to inhibit both B and T cell activation (1,2). In addition, NK cells express several HLA class I-specific receptors (NKR) which inhibit NK cell function upon interaction with their specific ligand (3,4). Some of these receptors belong to the Ig superfamily and recognize HLA-C (p58) (3), HLA-B (p70) (5,6) or HLA-A (p140) (7,8) alleles. Another receptor, characterized by a broader HLA class I specificity, is represented by CD94, a type II membrane protein belonging to the C-type lectin superfamily (9–11).

Recently, activating forms have been detected for both types of NKR. Cross-linking of these activating forms leads to NK cell triggering with intracellular calcium mobilization, cytokine production and target cell lysis (12,13). We have recently described that a novel surface molecule, termed p40, is able to inhibit the cytolytic activity mediated by both CD3–CD161 NK and CD3–TCR1 T cells against tumor target cells (14). The aim of this study was to define whether p40 molecule can also regulate activation signals and effector cell functions mediated by either activating NKR or CD3–TCR complex.

Correspondence to: A. Poggi Transmitting editor: J. F. Bach

Received 19 March 1997, accepted 26 May 1997

1272 Regulation of T and NK cell activation We found that NK cell-mediated lysis, cytokine release and calcium mobilization induced by cross-linking of activating NKR were strongly inhibited by anti-p40 mAb. In addition, the p40 molecule appears to regulate T cell triggering mediated by TCR engagement. Methods Reagents and mAb The anti-p58.2 (NKR for HLA-Cw3 alleles) mAb GL183 (IgG1) (15), the anti-p58.1 (NKR for HLA-Cw4 alleles) mAb EB6 (IgG1) (16), the anti-CD94 (NKR for HLA-Bw6 alleles) mAb XA185 (IgG1) (9), the anti-CD16 mAb VD4 (IgG1) (17), the anti-p40 mAb NKTA255 (IgG1) (14) and the anti-CD44 mAb T61/7 (IgG1) (18) were obtained in our laboratory. The antiTCR Vδ1 mAb (A13, IgG1), the anti-TCR Vδ2 mAb (BB3, IgG1) and the anti-TCR Vβ8 mAb (127/43, IgG2a) were produced in our laboratory as previously described (19–21). Anti-CD11a [lymphocyte function associated antigen (LFA)-1α] mAb TS1.22 (IgG1), anti-CD18 (LFA-1β) TS1.18 (IgG1) and antiCD45 mAb 9.4 (IgG2a) were from ATCC (Rockville, MD). AntiCD3 (Leu4a, IgG1), anti-CD4 (Leu3a, IgG1), anti-CD8 (Leu2a, IgG1), anti-CD56 (Leu19, IgG1) and anti-CD45RA (Leu18) (IgG1) were purchased from Becton Dickinson (Palo Alto, CA). The anti-CD3 mAb (UCHT-1, IgG1) was a kind gift from P. L. C. Beverly (Imperial Cancer Research Fund, London, UK). Phytohemagglutinin (PHA), acetoxymethyl ester of Fura-2 and EGTA were from Sigma (St Louis, MO). The affinity purified anti-Ig(H 1 L) (Fab9)2 goat anti-mouse serum (GAM) was purchased from Zymed (San Francisco, CA). All cells used in our experiments were cultured in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 10% of FCS (Biochrom) and with glutamine and penicillin–streptomycin (Biochrom). FITC-conjugated anti-mouse IgG1 or anti-mouse IgG2a goat anti-sera were from Southern Biotechnology (Birmingham, AL). Indirect immunofluorescence and cytofluorimetric analysis Single fluorescence staining was performed as described elsewhere (14). Briefly, aliquots of 13105 were stained with the corresponding mAb followed by FITC-conjugated antiisotype-specific GAM serum (Southern Biotechnology). Control aliquots were stained with the fluorescent reagent alone. Samples were analyzed on a flow cytometer (FACSort; Becton Dickinson) equipped with an argon ion laser exciting fluorescein at 488 nm. Data were analyzed using Lysys II (version 1.1). Calibration was assessed with CaliBRITE particles (Becton Dickinson) using the AutoCOMP computer program (version 2.1.2). Isolation and culture of CD3–CD161 NK cells and selection of NK cell clones Peripheral blood mononuclear cells (PBMC) from normal volunteers were isolated by Ficoll-Hypaque gradient. Adherent cells were subsequently eliminated by plastic adherence in a Petri dish for 2 h at 37°C. Highly purified CD3–CD161 NK cells were obtained from peripheral blood lymphocytes after depletion of CD31, CD41 and CD81 cells after incubation with anti-CD3 (Leu4a), anti-CD4 (Leu3a) and anti-CD8 (Leu2a)

mAb followed by immunomagnetic beads (Unipath), as described (14,22). Highly purified CD3–CD161 were stimulated with 10 µg/ml of PHA and then cultured in 96-well U-bottom microplates (Greiner Labortechnik, Nurtingen, Germany) with RPMI 1640 medium supplemented with 10% of FCS in the presence of 100 U/ml of rIL-2 (Cetus, Emeryville, CA) in a final volume of 200 µl/well. Under these culture conditions in 15 days virtually all proliferating cells expressed CD16 and CD56 antigens. CD3–CD161 NK cells clones were obtained by culturing highly purified CD3–CD161 NK cells under limiting dilution conditions as previously reported (9,22). All the NK cell clones were analyzed for the expression of the various NKR using specific mAb (GL183, EB6 and XA185). Only NK cell clones which homogeneously expressed the different NKR were used in functional assays. Generation of CD31 TCR1 T cell clones T cells TCRαβ1 CD81 Vβ81 were obtained from peripheral blood lymphocytes after depletion of CD41CD161 and enrichment in Vβ81 cells, whereas TCRγδ1 cells were isolated after depletion of CD41 CD81CD161. T lymphocytes were then cultured under limiting dilution conditions with 10 µg/ml of PHA and, after 24 h, rIL-2 was added at the final concentration of 50 U/ml. Cytolytic assay Cytolytic activity of CD3–CD161 NK cell clones or of CD31 TCRαβ1 or TCRγδ1 T cell clones was tested in a 4 h 51Cr-release assay as previously described in detail (14,23). CD3–CD161 NK cell clones were used as effector at an E:T ratio of 1:1 in a final volume of 200 µl of RPMI 1640, whereas TCRαβ1 Vβ81 T cell clones were used as effector at an E:T ratio of 20:1 and TCRγδ1 at an E:T ratio of 2:1. These clones were tested in a redirected killing assay using the murine mastocytoma cell line P815 (which express the receptor for the Fc fragment of Ig, FcγR1). Activation of cytolytic activity of CD3–CD161 NK cell clones was performed by adding saturating amounts (1–3 µg/106 cells) of the corresponding mAb (anti-NKR or anti-CD16) at the onset of the cytolytic assay. Activation of cytolytic activity of CD31 TCRαβ1 or TCRγδ1 T cell clones was performed by adding saturating amounts (1–3 µg/106 cells) of the corresponding mAb (antiCD3, anti-Vβ8 or anti-Vδ1 or anti-Vδ2 mAb) at the onset of the cytolytic assay. Inhibition experiments were performed by adding mAb recognizing p40 molecule, CD45, CD44, CD56, CD11a (LFA-1α) or CD18 (LFA-1β) at the onset of cytolytic assay together with stimulatory mAb. Determination of intracellular free calcium concentration ([Ca21]i) Determination of [Ca21]i was performed as previously described (22). Briefly, CD3–CD161 NK cell clones or CD31 TCRαβ1 or TCRγδ1 T cell clones were loaded with the acetoxymethyl ester of Fura-2 (1 µM final concentration, Sigma) at the concentration of 2.53106 cell/ml of the calcium assay buffer (22) and incubated for 45 min at 37°C. Subsequently, after two washes, NK cell clones were incubated for 30 min at 4°C with anti-NKR mAb (GL183 or XA185) in the presence or absence of anti-p40 mAb (NKTA255) or control mAb of the same isotype including anti-CD44 (T61/

Regulation of T and NK cell activation

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Table 1. Selection of NK cell clones bearing activating NKR for HLA class I antigens NK cell clonea

E11.100 H6.25 E12.100 E10.100 C5.100 E12.50 B10.100

Stimulus addedb None

Anti-CD16 mAb (VD4)

Anti-NKR for HLA-Cw3 mAb (GL183)

Anti-CD94 mAb (XA185)

3c 3 5 10 5 3 3

75 55 45 55 62 60 70

3 2 5 3 38 58 3

4 2 5 4 4 92 40

aA panel of CD3–CD161 NK cell clones expressing NKR for Cw3 HLA class I allele and CD94 were tested in a 4 h redirected killing assay using P815 target cells at an E:T ratio of 1:1. bCytolytic assay was performed after the addition of either medium (indicated with ‘none’) or mAb specific for the indicated molecules at saturating doses (1–3 µg/ml). cPercent of 51Cr-specific release.

Table 2. Engagement of p40 molecules inhibits NKR-mediated cytolytic activity NK cell clonea

Nonec Anti-p40 mAb (NKTA255) Anti-CD56 mAb (Leu19) Anti-LFA-1α (TS1.22) Anti-LFA-1β (TS1.18) Anti-CD45(9.4)


Anti-CD16 mAb (VD4)



5d 1 5 3 2 5

100 40 100 100 90 90

95 35 100 95 90 90

82 10 84 80 80 70

aThe CD3–CD161 NK cell clone B3.50 expressing NKR for HLA-Cw3 alleles and CD94 molecule was tested in a 4 h redirected killing assay using P815 target cells at an E:T ratio of 1:1. bCytolytic assay was performed after the addition of either medium (indicated with ‘none’) or mAb specific for the indicated molecules at saturating doses (1–3 µg/ml). cInhibition of either CD16- or NKR-mediated cytolytic activity was performed by adding saturating amounts (1–3 µg/ml) of the mAb specific for the indicated surface molecules. ‘None’ corresponds to cytolytic activity in the absence of mAb. dPercent of 51Cr-specific release. Similar results were obtained with three different NK cell clones displaying an identical surface phenotype and functional characteristics.

7), anti-CD56 (Leu19), anti-CD45RA (Leu18) or anti-CD8 (Leu2a) mAb. T cell clones CD31 TCRαβ1 and CD31 TCRγδ1 were incubated respectively with anti-CD3 (UCHT-1) or antiVβ8(127/43) mAb or with anti-TCRγδ (A13 or BB3) mAb, in the presence or in the absence of anti-p40 mAb (NKTA255). After two washes the fluorescence of the cellular suspension was monitored with an LS-50 spectrofluorimeter (Perkin-Elmer, Ponoma, CA) using a 2 ml quartz cuvette. The cell suspension was excited at 340–380 nm and fluorescence emission was measured at 510 nm. All measurements were performed at 37°C using a thermostatically controlled cuvette holder and stirring apparatus as described (22). [Ca21]i was calculated by the method of Grynkiewicz et al. (24). Oligomerization of the molecules pretreated with the various mAb was obtained by adding a large excess of (20 µg/ml) (determined in preliminary experiments) (Fab9)2 polyclonal serum directed against mouse IgG Ig (GAM) (22). Calcium mobilization from internal stores was evaluated by adding EGTA (Sigma) in order to avoid calcium entry, whereas the opening of membrane calcium channels was evaluated by adding an excess

of CaCl2 (1 mM) to extracellular medium after stimulation of NK cells in the presence of EGTA. Determination of IFN-γ and tumor necrosis factor (TNF)-α production CD3–CD161 NK cell clones were pretreated with anti-CD16 or anti-NKR mAb, either alone or in the presence of the antip40 mAb and added to 24-well flat bottom Costar plates coated with 20 µg/ml of GAM to obtain optimal cross-linking of corresponding molecules. After 24 h, the culture supernatants were harvested and the amount of IFN-γ and TNF-α was quantitated by either biological assays (25,26) or using immunoenzymatic assay kits (Medgenix, Fleurus, Belgium). The IFN-γ and TNF-α concentrations were determined using a Gralis Automatic Microplate Reader supported with Medgenix Elisa-Aid software. Proliferation assay PBMC were cultured in complete medium in 200 µl/105 cells/ well in 96 U-bottomed microplates in the presence or absence

1274 Regulation of T and NK cell activation Table 3. The engagement of p40 molecules inhibits NKR-mediated [Ca21]i increase. NK cell clonea

Nonec Anti-p40 mAb (NKTA255) Anti-CD44 mAb (T61/7)


Anti-CD16 mAb (VD4)



5d 5 7

800 270 790

650 280 660

850 170 840

aThe CD3–CD161 NK cell clone B1.100 expressing NKR for HLA-Cw3 alleles and CD94 molecule was tested in a calcium mobilization assay as described in Methods. bNK cells were pretreated with saturating amounts (1–3 µg/ml) of the indicated mAb for 30 min at 4°C, washed and used in the calcium mobilization assay. cNK cells were pretreated with saturating amounts of either anti-p40 mAb (NKTA255) or anti-CD44 mAb (T61/7) (1–3 µg/ml). ‘None’ corresponds to [Ca21]i increase after the addition of 20 µg/ml of (Fab9)2 GAM. dResults are expressed as nM [Ca21] above basal level after the addition of 20 µg/ml of (Fab9) GAM. Similar results were obtained i 2 analyzing four different NK cell clones with the same surface phenotype and functional characteristics.

Fig. 1. The anti-p40 mAb NKTA255 inhibits the [Ca21]i increase mediated by NKR engagement. The NK cell clone B3.50 labeled with Fura-2 was incubated with medium (A) or with saturating amounts of the anti-CD56 mAb (Leu19) (B) or the anti-NKR for HLA-Cw3 mAb (GL183) alone (C) or with either anti-p40 mAb (NKTA255) (D) or anti-CD44 mAb (T617) (E) for 30 min at 4°C. The cross-linking of these molecules was achieved by the addition of 20 µg/ml of F(ab9)2 GAM and [Ca21]i increase was measured according to Grynkiewicz et al. (24). The depletion of extracellular calcium was obtained by adding 5 mM EGTA (C–E) prior the addition of F(ab9)2 GAM. To determine whether opening of calcium channels at the cell surface has occured after the cross-linking of NKR molecules an excess of CaCl2 (10 mM) was added when [Ca21]i was reverted at the basal level. Results are expressed as nanomolar [Ca21]i and are representative of data obtained with six different NK cell clones.

of anti-Vβ8 mAb (127/43) or anti-Vδ2 mAb (BB3) alone in the presence of either anti-p40 mAb (NKTA255) or anti-CD44 (T61/7) for different periods of time at 37°C in 5% CO2 atmosphere. In some experiments, PBMC-derived CD3– CD161 NK cells were cultured with 100 IU/ml of rIL-2 alone or in the presence of anti-p40 mAb (NKTA255) or antiCD44 mAb (T61/7). Under these experimental conditions cells recovered on day 5 were represented by 90% of CD161CD561 NK cells. Cell proliferation was assessed after labeling with 20 µCi [3H]thymidine and additional incubation for 18 h at 37°C. Results are expressed in c.p.m.310–3 of mean 6 SD of triplicate samples.

Results and discussion The p40 molecule regulates the NKR-mediated cytolytic activity CD3–CD161 NK cell clones were selected for the expression of NKR for HLA class I molecules by indirect immunofluorescence and FACS analysis (not shown). We selected clones which reacted with GL183 mAb (recognizing NKR for HLACw3) and XA185 mAb (recognizing CD94 molecule). All the clones were then tested in a redirected killing assay against the murine mastocytoma cell line P815 in the presence of specific mAb directed to NKR molecules. Among the clones


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gesting that LFA-1 and CD45 molecules are not involved in the regulation of NKR-mediated activation of NK cells. Altogether these findings suggest that the p40 molecule regulates NK cell activation induced by NKR engagement and that this effect is likely to be independent of either CD45 or LFA-1-mediated activities. Oligomerization of the p40 molecule inhibits the [Ca21]i increase induced by NKR engagement

Fig. 2. p40 molecule oligomerization inhibits TNF-α and IFN-γ production induced by NKR engagement. The NK cell clone B1.100 was preincubated with the indicated mAb for 30 min at 4°C, washed and cultured on 24-well Costar plates coated with 20 µg/ml of F(ab9)2 GAM for 24 h. Culture supernatants were evaluated for TNF-α (A) or IFN-γ (B) production by immunoenzymatic assay. NK cells were q) or in combination preincubated with anti-CD16 mAb alone (VD4) (k with anti-p40 (NKTA255) mAb (m); anti-NKR for HLA-Cw3 mAb (GL183) alone (r) or in combination with anti-p40 (NKTA255) mAb (u); anti-CD94 mAb (XA185) alone ( ) or in combination with antip40 mAb (NKTA255) (u). ‘None’ indicate cells incubated with medium alone. Results are expressed as pg/ml of TNF-α or IU/ml of IFN-γ, and are representative of four different NK cell clones tested.

analyzed, a minority (5–10%) expressed activating NKR based on the finding that addition of anti-NKR mAb induced a strong increase of cytolytic activity (Table 1). We previously reported that mAb directed to the p40 molecule strongly inhibited target cell lysis mediated by NK cells upon activation via CD16 molecules (14). Since CD16 molecule represents one of the main activation pathways of NK cells, we analyzed whether anti-p40 mAb could affect NKR-mediated activation as well. To this aim, the CD3–CD161 NK cell clones selected as described above were used in a redirected killing assay against the P815 target cell line. As shown in Table 2, the cytolytic activity induced by mAb recognizing different NKR was strongly inhibited by the addition of the anti-p40 mAb. Control mAb recognizing other molecules expressed on the NK cell surface including CD56 (Table 2) and CD44 (data not shown) did not exert any inhibitory effect. It is well known that the tyrosine phosphatase CD45 and the adhesion molecule LFA-1 are able to regulate NK cell-mediated cytolytic activity (22,27–29). Thus, redirected killing experiments in the presence of mAb recognizing either CD45 or LFA-1 were performed. As shown in Table 2, anti-CD45 or anti-LFA-1 mAb did not inhibit NK cell-mediated cytolysis induced by either anti-NKR or CD16 mAb, sug-

We further analyzed whether the cross-linking of p40 molecules could affect NKR-mediated early activation signals. To this aim, CD3–CD161 NK cell clones expressing activating NKR were pretreated with anti-NKR and anti-p40 mAb followed by the addition of (Fab9)2 GAM. As shown in Table 3, oligomerization of p40 molecules strongly inhibited NKR-mediated [Ca21]i increase. Simultaneous oligomerization of NKR and other molecules, including CD44 (Table 3), CD8 and CD45RA (not shown) (which were expressed at comparable levels to p40 molecules, not shown) did not affect the [Ca21]i increase mediated by NKR cross-linking. In another series of experiments, we investigated whether the p40-mediated regulation of [Ca21]i increase elicited via NKR affected either calcium mobilization from internal stores or calcium influx from the extracellular medium. As shown in Fig. 1(C), in the presence of EGTA (which depletes extracellular Ca21), cross-linking of NKR induced a strong and transient [Ca21]i increase. Moreover, the addition of CaCl2 when [Ca21]i was reverted at basal level led to a further calcium mobilization due to calcium influx from extracellular medium. These findings indicate that NKR-mediated [Ca21]i increase was dependent both on calcium mobilization from internal stores and opening of calcium channels at the cell surface. In control experiments, the addition of (Fab9)2 GAM to either untreated (Fig. 1A) or anti-CD56 mAb treated cells (Fig. 1B) did not induce any [Ca21]i increase. The simultaneous cross-linking of NKR and p40 molecules reduced both calcium mobilization from internal stores and extracellular calcium influx (Fig. 1D). On the contrary, no effect was observed by simultaneous oligomerization of NKR and CD44 (Fig. 1E). Altogether, these findings indicated that p40 molecules regulate the early activation signals elicited via cross-linking of NKR molecules. The p40 molecule engagement inhibits NKR-mediated IFN-γ and TNF-α production It has been demonstrated that activation of CD3–CD161 NK cell clones via CD16 or activating NKR for HLA-class I can induce the production of cytokines such as IFN-γ and TNF-α (12,13). In this context, we investigated whether the engagement of p40 molecules could regulate this NKR-mediated effector cell function as well. To this aim, some CD3–CD161 NK cell clones bearing activating NKR were stimulated with anti-NKR mAb, in the presence or in the absence of anti-p40 mAb. Cell culture supernatants were analyzed for the content of TNF-α and IFN-γ after 24 h. As shown in Fig. 2, the crosslinking of p40 and CD94 molecules consistently led to a strong inhibition of CD94-mediated TNF-α (Fig. 2A) and IFN-γ (Fig. 2B) production. In addition, cross-linking of either LFA-3 or CD44 with NKR molecules did not modify the NKR-mediated TNF-α and IFN-γ production (not shown). It should be noted that when p40 and NKR for HLA-Cw3 molecules were simul-

1276 Regulation of T and NK cell activation

Fig. 3. The p40 molecule engagement inhibits TCR-mediated cytolytic activity. TCR-mediated cytolytic activity of polyclonal or clonal T cell populations was tested in a redirected killing assay using P815 target cells. (A and B) Two CD81 Vβ81 bulk populations were incubated with P815 at the E:T ratio of 20:1, in the presence of anti-p40 mAb (NKTA255) (k q); or anti-CD3 mAb (UCHT-1) alone (m) or with anti-CD3 plus anti-p40 mAb (r); or anti-Vβ8 mAb (127/43) alone (u) or with anti-Vβ8 plus anti-p40 mAb (w). ‘None’ indicates cells incubated in medium alone (j). (C–F) Cytolytic activity of Vβ81 T cell clones (A7.5 and D4.10) (C and D) or Vδ11 or Vδ21 T cell clones (15 and 67) (E and F) against q), anti-CD3 mAb alone (m), anti-CD3 and anti-p40 mAb (r), anti-TCR mAb P815 target cells was analyzed in the presence of anti-p40 mAb (k [anti-Vβ8 mAb (127/43) in panels (C) and (D), anti-Vδ2 (BB3) mAb in panel (E), and anti-Vδ1 mAb (A13) in panel (F)] (u) alone or with antip40 mAb ( ). ‘None’ indicates cells incubated in medium alone (j). Results are expressed as specific 51Cr release. Similar results were obtained using 20 Vβ81 cell clones and 12 Vδ1 T cell clones.

taneously engaged no inhibitory effect was detectable in clone B1.100 (Fig. 2) as well as in three other NK cell clones. However, cytolytic activity and intracellular calcium increases induced by NKR cross-linking in these clones were inhibited by p40 molecule engagement. These different inhibitory effects could be due to the experimental conditions used in these functional assays. In fact, it is conceivable that the degree of molecule cross-linking achieved by the addition of GAM is different when added in a soluble or immobilized form. The different susceptibility of CD94- and NKR for HLA-Cw3-mediated lymphokine production to p40 molecule

inhibitory effect may be dependent on the downstream activation signals involved by the engagement of these NKR. Effect of p40 molecule engagement on CD3–TCR-mediated T cell triggering As the p40 molecule is expressed not only on NK cells, but also on T lymphocytes, we investigated whether this molecule also regulates TCR-mediated T lymphocyte triggering. To this aim T cell clones expressing either TCRαβ or TCRγδ were tested in a redirected killing assay using anti-TCR-specific mAb in the presence or in the absence of anti-p40 mAb. As


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Fig. 4. The p40 molecule engagement regulates TCR Vβ8-mediated Ca21 mobilization. The T cell clone A7.5 Vβ81 (representative of five TCRαβ1 clones tested) was labeled with Fura-2 and then incubated with anti-CD3 mAb (UCHT1) or with anti-CD3 and anti-p40 mAb (UCHT1 plus NKTA255) (A), or with anti-Vβ8 mAb (127/43) or with anti-Vβ8 and anti-p40 mAb (127/43 plus NKTA255) (B) for 30 min a 4°C. The T cell clone 15 Vδ21 (representative of four TCRγδ1 clones tested) was pretreated with anti-Vδ2 mAb (BB3) or with anti-Vδ2 and anti-p40 mAb (BB3 plus NKTA255) (C). The cross-linking of these molecules was achieved by the addition of 20 µg/ml of F(ab9)2 GAM and calcium increase was measured according to Grynkiewicz et al. (24). Results are expressed as nanomolar [Ca21]i.

Fig. 5. Anti-p40 mAb induces inhibition of T and NK cell proliferation. PBMC was cultured in the presence of mAb directed to TCRαβ or γδ (u) [A: anti-Vβ8 mAb (127/43); B: anti-Vδ2 mAb (BB3)] alone or in combination with anti-p40 (NKTA255) (j) or with anti-CD44 (T61/7) (r) mAb. In (C), PBMC-derived CD3–CD161 NK cells were cultured with IL-2 100 IU/ml alone (u) or in combination with anti-p40 mAb (j) or with anti-CD44 mAb (r). ‘None’ indicates cells incubated with medium alone. Cell proliferation was evaluated by [3H]thymidine uptake at day 5 of culture. Results are expressed as c.p.m.310–36 SD of triplicate samples. This experiment is representative of five performed.

shown in Fig. 3, the engagement of the p40 and Vβ8 molecule strongly inhibited (by 50–70%) the cytolysis of P815 target cells exerted by two different Vβ81 bulk populations (Fig. 3A and B). At the clonal level, the p40-mediated inhibitory effect on TCR-mediated activation was evident only in 50% of Vβ81 clones tested. In fact, a slight inhibition (by 10%) of the cytolysis of P815 target cells was found using the D4.10 Vβ81 T cell clone (Fig. 3C), whereas a stronger inhibition (by 50%) was detectable using the A7.5 Vβ81 T cell clone (Fig. 3D). On the other hand, the cross-linking of p40 and TCR molecules on either Vδ11 or Vδ21 T cell clones led to a weak inhibition ranging from 5 to 20% (Fig. 3F and E). As previously reported (14), the cytolytic activity of either TCRαβ1 or TCRγδ1 T cell clones triggered using anti-CD3 mAb was not altered by the cross-linking of p40 and CD3 molecules (Fig. 3). It should be noted that the stimulation of T cell clones with anti-CD3 mAb led to a stronger activation (from 30 to 50%) than that elicited by anti-TCR specific mAb in the large majority of clones tested. Furthermore, as shown in Fig. 4, the p40 engagement

together with Vβ8 molecules on Vβ81 T cell clones led to a detectable inhibition (30%) (Fig. 3B) of TCR-mediated [Ca21]i increase. By contrast, the cross-linking of p40 with either CD3 or Vδ2 molecules had no effect (Fig. 3A and C). This different inhibitory effect on T cell clones belonging to different T cell subset was not dependent on the level of expression of p40 molecules. In fact, both TCRαβ1 or TCRγδ1 activated T cells display a similar amount of p40 molecules at the cell surface (not shown). Altogether, these findings suggest that the p40 molecules may play a role in the regulation of activation of TCRαβ1 T cell clones. The p40 molecule regulates both NK and T cell proliferation In another series of experiments, we also investigated whether the p40 molecule is involved in the regulation of TCR-mediated activation of resting T lymphocytes and NK cell proliferation to exogenous IL-2. To this aim PBMC were stimulated with anti-TCR-specific mAb (anti-Vβ8 or anti-Vδ2 mAb) in the presence or absence of anti-p40 mAb and proliferation was

1278 Regulation of T and NK cell activation assessed at different time intervals from day 3 to day 6. The maximal TCR-mediated T cell proliferation was observed on day 5. As shown in Fig. 5, proliferation of TCRαβ1 (Fig. 5A) as well as that of TCRγδ1 lymphocytes (Fig. 5B) was strongly inhibited by the addition of anti-p40 mAb. On the other hand, the engagement of TCR with CD44 (Fig. 5) or CD58 (data not shown) did not exert any detectable effect. Furthermore, the p40 molecule engagement led to a strong inhibition of NK cell proliferation to exogenous IL-2 (Fig. 5C). Although not shown, NK cells cultured with IL-2 in the presence of antip40 mAb did not acquire the ability of lysing fresh tumor target cells. Altogether these findings suggest that p40 molecule is involved in the regulation of proliferation and functional maturation of both T and NK lymphocytes.

that the p40 molecule could modulate TCR-mediated T cell responses such as T cell proliferation to (allo)antigens. The degree of the p40-mediated inhibition of TCR-induced cytolysis was not similar to that observed on NKR-mediated activation of NK cells. This was not dependent on a different expression of the p40 molecule on activated T versus activated NK cells. Experiments are in progress in order to define whether the p40 molecule is associated to different surface structures on T and NK cells. In conclusion, our present data support the concept that the p40 molecule may play a regulatory role in the maturation of T and NK effector cells as well as in T and NK cell-mediated functional activities. Acknowledgements

Concluding remarks Here, we have shown that activation of NK cells and T lymphocytes initiated by activating NKR or TCR engagement respectively is regulated by cross-linking of the recently described p40 molecule (14). Indeed, the engagement of the p40 molecule with activating NKR on NK cells led to a strong inhibition of cytolytic activity, calcium mobilization and production of inflammatory lymphokines as TNF-α and IFN-γ induced by NKR alone. Activating NKR recognize the same HLA supertypic alleles as inhibitory NKR, but this recognition would result in the activation of NK cell-mediated cytolysis (12,13). Thus, the presence of these activating receptors at the NK cell surface would lead to lysis of autologous cells. The p40mediated inhibitory effects would represent a mechanism by which autologous cell lysis is down-regulated. In this context, the decrease, mediated by p40 molecule engagement, in the amount of inflammatory lymphokines produced by NK cells upon stimulation initiated via activating NKR molecules would accompany the inhibition of cytolysis. This would further diminish the undesirable effects elicited by NKR activation. The inhibition of NKR-mediated [Ca21]i increase induced by p40 molecule cross-linking would indicate that the engagement of the p40 molecule interferes with NKR-mediated early signal transduction. Experiments aimed to further analyze this point are in progress in our laboratory. Finally, one could hypothesize that the binding of the p40 molecule with its physiological ligand expressed by autologous cells downregulates the NKR-mediated signaling, blocking lysis of autologous cells. The finding that neither CD45 nor LFA-1 appeared to affect activating NKR-mediated target cell lysis would imply that these two surface molecules [which play a major role in regulating spontaneous NK cell-mediated cytolytic activity (27–29)] are not involved in the regulation of activating NKR-dependent activation. The cross-linking of the p40 molecule could also inhibit TCR-mediated T lymphocyte activation. However, the p40mediated inhibitory effect was detectable in TCR αβ1 but not in TCRγδ1 T cell clones. This difference could be due to the different activation threshold in these T lymphocytes subpopulations after culture with rIL-2. This interpretation was supported by the finding that TCR-induced proliferation of freshly isolated peripheral blood T lymphocytes expressing either TCR αβ1 or TCRγδ1 was inhibited at the same extent by the engagement of p40 molecules. Further, this suggests

This work was partially supported by grants awarded by: Associazione Italiana per la Ricerca sul Cancro (AIRC), Istituto Superiore di Sanita` (ISS), Consiglio Nazionale delle Ricerche (CNR), Progetto Finalizzato ACRO. E. T. is a recipient of a fellowship awarded by AIRC.

Abbreviations GAM LFA NKR PBMC PHA TNF

goat anti-mouse lymphocyte function associated antigen NK receptor for HLA class I peripheral blood mononuclear cell phytohemagglutinin tumor necrosis factor

References 1 Muta, T., Kurosaki, Z., Misulovin, M., Sanchez, M., Nussenzweig, M. C. and Ravetch, J. V. 1994. A 13-aminoacid motif in the cytoplasmic domain of FC gamma RIIB modulates B-cell receptor signaling. Nature 368:70. 2 D’Ambrosio, D., Hippen, K. L., Minskoff, S. A., Mellman, I., Pani, G., Simonovitch, K. A. and Cambier, J. C. 1995. Recruitment and activation of PTP1C in negative regulation of antigen receptor signaling by FcgammaRIIB1. Science 268:293. 3 Moretta, A., Bottino, C., Vitale, M., Pende, D., Morelli, L., Augugliaro, R., Bottino, C. and Moretta, L. 1996. Receptors for HLA-I molecules in human Natural Killer cells. Annu. Rev. Immunol. 14:619. 4 Lanier, L. L. and Phillips, J. H. 1996. Inhibitory MHC class I receptors on NK cells and T cells. Immunol Today 17:86. 5 Litwin, V., Gumperz, J. E., Parham, P., Phillips, J. H. and Lanier, L. L. 1994. NKB1: a natural killer cell receptor involved in the regulation of polymorphic HLA-B molecules. J. Exp. Med. 180:537. 6 Vitale, M., Sivori, S., Pende, D., Augugliaro, R., Di Donato, C., Amoroso, A., Malnati, M., Bottino, C., Moretta, L. and Moretta, A. 1996. Physical and functional independency of p70 and p58 natural killer (NK) cell receptors for HLA class I: their role in the definition of different groups of alloreactive NK cell clones. Proc. Natl Acad. Sci. USA 93:1453. 7 Dohring, C., Scheidegger, D., Samaridis, J., Cella, M. and Colonna, M. 1996. A human killer inhibitory receptor specific for HLA-A. J. Immunol. 156:3098. 8 Pende, D., Biassoni, R., Cantoni, C., Verdiani, S., Falco, M., di Donato, C., Accame, L., Bottino, C., Moretta, A. and Moretta, L. 1996. The natural killer cell receptor specific for HLA-A allotypes. A novel member of the p58/p70 family of inhibitory receptors which is characterized by three Ig-like domains and is expressed as a 140kD disulphide-linked dimer. J. Exp. Med. 184:1. 9 Lopez-Botet, M. 1995. CD94 Cluster report: overwiev of the characterization of Kp43 NK-cell associated surface antigen. In Schlossman, S. F., Boumsell, L., Gilks, W., Harlan, J. H., Kishimoto, T., Morimoto, C., Ritz, J., Shaw, S., Silverstein, R., Springer, T.,


10 11

12

13

14

15

16

17

18

19

Tedder, T. F. and Todd, R. F., eds, Leucocyte Typing V, p. 1437. Oxford University Press, Oxford. Moretta, A., Vitale, C., Sivori, S., Morelli, L., Pende, D. and Bottino, C. 1996. Molecular basis of NK cell recognition and function. Chem. Immunol. 64:77. Phillips, J. H., Chang, C., Mattson, J., Gumperz, J. E., Parham, P. and Lanier, L. L. 1996. CD94 and a novel associated protein (CD94AP) form a NK cell receptor involved in the recognition of HLA-A, HLA-B and HLA-C allotype. Immunity 5:163. Moretta, A., Sivori, S., Vitale, M., Pende, D., Morelli, L., Augugliaro, R., Bottino, C. and Moretta, L. 1995. Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells. J. Exp. Med. 182:875. Pe´rez- Villar, J. J., Melero, I., Rodrı`guez, A., Carretero, M., Aramburu, J., Sivori, S., Orengo, A. M., Moretta, A. and Lo`pezBotet, M. 1995. Funtional ambivalence of the Kp43 (CD94) NK cell-associated surface antigen. J. Immunol. 154:5779. Poggi, A.,Pella, N., Morelli, L., Spada, F., Revello, V., Sivori, S., Augugliaro, R., Moretta, L. and Moretta, A. 1995. p40, a novel surface molecule involved in the regulation of the non-major histocompatibility complex-restricted cytolytic activity in humans. Eur. J. Immunol. 25:369. Moretta, A., Tambussi, G., Bottino, C., Tripodi, G., Merli, A., Ciccone, E., Pantaleo, G. and Moretta, L. 1990. A novel surface molecule antigen expressed by a subset of human CD3–CD161 natural killer cells. Role in cell activation and regulation of cytolytic function. J. Exp. Med. 71:695. Moretta, A., Bottino, C., Pende, D., Tripodi, G., Tambussi, G., Viale, O., Orengo, A., Barbaresi, M., Merli, A., Ciccone, E. and Moretta, L. 1990. Identification of four subset of human CD3– CD161 natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK cell clones and ability to mediate specific alloantigen recognition. J. Exp. Med. 172:1589. Moretta, A., Tambussi, G., Ciccone, E., Pende, D., Melioli, G. and Moretta, L. 1989. Surface molecules regulate the cytolytic function of CD3–CD161 human ‘natural killer’ cells. Int. J. Cancer 44:727. Zocchi, M. R., Vidal, M. and Poggi, A. 1993. Involvement of N-CAM molecule in the adhesion of human solid tumor cell line to endothelial cells. Exp. Cell. Res. 204:130. Bottino, C., Tambussi, G., Ferrini, S., Ciccone, E., Mingari, M. C., Varese, P., Moretta, L. and Moretta, A. 1988. Two subset of human

20

21

22

23

24 25

26 27 28 29

1279

T lymphocytes expressing gamma–delta antigen receptor are identifiable by monoclonal anti-bodies directed to two distinct molecular forms of the receptor. J. Exp. Med. 168:491. Ciccone, E., Ferrini, S., Bottino, C., Viale, O., Prigione, I., Pantaleo, G., Tambussi, G., Moretta, A. and Moretta, L. 1988. A monoclonal antibody specific for a common determinant of the human T cell receptor γδ directly activates CD31 WT31– lymphocytes to express their functional program(s). J. Exp. Med. 168:1. Moretta, A., Poggi, A., Olive, D., Bottino, C., Pantaleo, G., Fortis, C. and Moretta, L. 1987. Selection of characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44 and T11): analysis of the functional relationship among different pathways of activation. Proc. Natl Acad. Sci. USA 84:1654. Poggi, A., Pardi, R., Pella, N., Morelli, L., Spada, Sivori, S., Vitale, M., Revello, V., Moretta, A. and Moretta, L. 1993. CD45-mediated regulation of LFA1 function in human natural killer cells. AntiCD45 monoclonal anti-bodies inhibit the calcium mobilization induced via LFA1 molecules. Eur. J. Immunol. 23:2454. Moretta, A., Pantaleo, G., Moretta, L., Cerottini, J. C. and Mingari, M. C. 1983. Direct demonstration of the clonogenic poential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity. J. Exp. Med. 157:743. Grynkiewicz, G., Poemie, M. and Tsien, R. Y. 1985. A new generation of Ca21 indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440. Colotta, F., Bersani, L., Lazzarin, A., Poli, G. and Mantovani, A. 1985. Rapid killing of actinomycin D-treated tumor cells by human monocytes II. Cytotoxicity is independent of secretion of reactive oxygen intermediates and is suppressed by protease inhibitors. J. Immunol. 134:3524. Ferrarini, M., Ferrero, E., Fortis, C., Poggi, A. and Zocchi, M. R. 1990. LAK1 antigen defines two distinct subsets among human tumour infiltrating lymphocytes. Br. J. Cancer. 62:754. Thomas, M. L. 1989. The leukocyte common antigen family. Annu. Rev. Immunol. 7:339. Newman, W., Fast, L. D. and Rose, L. M. 1983. Blockade of NK cell lysis is a property of monoclonal anti-bodies that bind to distinct regions of T-200. J. Immunol. 131:1742. Seaman, W. E., Talal, N., Herzemberg, L. A. and Ledbetter, J. A. 1981. Surface antigens on mouse natural killer cells: use of monoclonal antibodies to inhibit or to enrich cytotoxic activity. J. Immunol. 127: 982.